Blood is a liquid tissue that includes red cells, white cells, corpuscles, and platelets dispersed in a liquid phase. The liquid phase is plasma, which includes acids, lipids, dissolved electrolytes, and proteins. One particular protein suspended in the liquid phase is fibrinogen. When bleeding occurs, the fibrinogen reacts with water and thrombin (an enzyme) to form fibrin, which is insoluble in blood and polymerizes to form clots.
In a wide variety of circumstances, animals, including humans, can suffer from bleeding due to wounds or during surgical procedures. In some circumstances, the bleeding is relatively minor, and normal blood clotting functions in addition to the application of simple first aid are all that is required. In other circumstances substantial bleeding can occur. These situations usually require specialized equipment and materials as well as personnel trained to administer appropriate aid.
In an effort to address the above-described problems, materials have been developed for controlling excessive bleeding.
The previously known materials, such as gelatin, collagen, oxidized cellulose, thrombin, fibrinogen, and other materials have been used, but each of these materials has limitations. For example, one type of prior art blood clotting materials are blood-derived proteins or enzymes, including fibrinogen and/or thrombin, which are expensive, need specialized storage conditions, and require extensive purification in order to eliminate the potential for transmission of blood-borne infections.
Hemostatic devices containing liquid thrombin have special handling requirements in order to maintain thrombin's biologic activity. For example, liquid thrombin requires refrigeration to maintain shelf-life stability. Safety is also a concern when using animal or human derived thrombin as there are some risks of contaminants or immunogenicity. Further, thrombin and fibrinogen purified from human or animal plasma are very expensive. Therefore, it is advantageous to develop novel hemostats with greater shelf-life stability, lower risk of viral etc. contaminants and lower immunogenicity, low cost, and which can work in heparinized blood.
Human protein Thrombospondin-1 (TSP-1) and related peptides were described in literature as to be involved in angiogenesis and platelet aggregation. TSP-1 is a homotrimeric glycoprotein (MW ˜450K) and was first discovered in platelets as a thrombin-sensitive protein.
An article “The evolving role of thrombospondin-1 (TSP1) in hemostasis and vascular biology” by Bonnefoy et al., Cell. Mol. Life Sci. 65 (2008) 713-727, describes TSP1 as involved in angiogenesis, inflammation, wound healing and hemostasis and further references the structure and domains of the TSP1 as well as the binding of the amino acid (SEQ ID NO: 1) peptide to CD47.
An article “Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 requires the docking protein LAT but is largely independent of alphaIIb/beta3”, by Trumel et al., Journal of Thrombosis and Haemostasis, 2003 February; 1(2):320-9, teaches that thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, SEQ ID NO: 1 can activate human platelets at least in part via its binding to integrin-associated protein.
An article “Thrombspondin Acts via Integrin-associated Protein to Activate the Platelet Integrin αIIbb3”, by Chung et al., J Biol Chem 1997; 272 No. 23, Issue of June 6, pp. 14740-14746, teaches that a peptide from the CBD, SEQ ID NO: 2 (4N1K) has been identified as an IAP agonist. TS1, the CBD, and an IAP agonist peptide (4N1K) from the CBD of TS1 activate the platelet integrin aIIbb3, resulting in platelet spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion kinase.
An article “Thrombospondin promotes platelet aggregation” by Tuszynski et al, Blood, 72, 109-115 (1988), teaches that while the role of TSP in hemostasis is not well understood, it has been postulated that TSP crosslinks platelet-fibrinogen aggregates, stabilizes fibrin clot formation, and modulates fibrinolysis and that a study has suggested that TSP may play a regulatory role in hemostasis by inhibiting platelet adhesion and providing a nonthrombogenic surface.
An article “Thrombospondin-1 acts via IAP/CD47 to synergize with collagen in alpha2beta1-mediated platelet activation”, by Chung et al., Blood. 1999 Jul. 15; 94(2):642-8 describes CD47 agonist peptide, 4N1K (SEQ ID NO: 2), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation.
An article “Stimulation of platelet activation and aggregation by a carboxyl-terminal peptide from thrombospondin binding to the integrin-associated protein receptor”, by Dorahy et al., J. Biol Chem 1997; 272:1323-1330, teaches that a peptide from the carboxyl terminus of thrombospondin, SEQ ID NO: 1, directly and specifically induces the activation and aggregation of washed human platelets from different donors at concentrations of 5-25 mM. At lower concentrations the peptide synergizes with suboptimal concentrations of ADP to induce aggregation. Peptide affinity chromatography and immunoprecipitation with a monoclonal antibody were used to identify the receptor for the carboxyl-terminal peptide as the integrin-associated protein. The integrin-associated protein remained bound to the SEQ ID NO: 1 containing peptide column when washed with a scrambled peptide in the presence of 5 mM EDTA, indicating a divalent cation-independent association. It is suggested that integrin-associated protein is the primary receptor for thrombospondin on the surface of resting platelets and is implicated in potentiating the platelet aggregation response.
An article “Thrombospondin-bound integrin-associated protein (CD47) physically and functionally modifies integrin alphaIIbbeta3 by its extracellular domain”, by Fujimoto et al., J Biol Chem 2003; 278:26655-26665, teaches that a peptide from the C-terminal cell binding domain, SEQ ID NO: 2 (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-D-glucose, although ADP or collagen-induced platelet response was completely inhibited.
An article “C-terminal peptide of thrombospondin-1 induces platelet aggregation through the Fc receptor chain-associated signaling pathway and by agglutination” by Tulasne et al., Blood 2001; 98:3346-3352, teaches that a peptide from the C-terminal domain of thrombospondin-1 (Arg-Phe-Tyr-Val-Val-Met-Trp-Lys (SEQ ID NO: 1); known as 4N1-1) has been reported to induce platelet aggregation and to bind to the integrin-associated protein (TAP), which is also known as CD47. It was discovered that 4N1-1 or its derivative peptide, 4N1K, induces rapid phosphorylation of the Fc receptor (FcR) g chain, Syk, SLP-76, and phospholipase C g2 in human platelets. The reference teaches that that the C-terminal peptide of thrombospondin induces platelet aggregation through the FcR g-chain signaling pathway and through agglutination.
An article “The C-terminal peptide of thrombospondin-1 stimulates distinct signaling pathways but induces an activation-independent agglutination of platelets and other cells,” by Voit et al., FEBS Letters, 2003; 544: 240-245 teaches that a peptide from the C-terminal domain of thrombospondin-1 (4N1-1) has been proposed to stimulate platelet aggregation by a novel mechanism involving both an activation-independent agglutination and an activation-dependent, glycoprotein (GP) IIb/IIIa-mediated aggregation which involves GPVI signaling but does not involve CD47. The study demonstrates that 4N1-1 stimulated a different pattern of signal transduction pathways than the GPVI agonist convulxin. Furthermore, 4N1-1-induced platelet aggregation was activation-independent and not dependent on GPVI or GPIIb/IIIa. 4N1-1 also stimulated activation-independent agglutination of different megakaryocytic and non-megakaryocytic cells. 4N1-1-induced cell agglutination but not platelet signaling was inhibited by anti-CD47 antibodies.
An article “A model of platelet aggregation involving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor” by Bonnefoy et al., J Biol Chem 2001; 276:5605-5612, teaches that Thrombospondin-1 (TSP) may, after secretion from platelet a granules, participate in platelet aggregation, but its mode of action is poorly understood. The study evaluated the capacity of TSP to form inter-platelet cross-bridges through its interaction with fibrinogen (Fg), using either Fg-coated beads or Fg bound to the activated GPIIbIIIa integrin (GPIIbIIIa*) immobilized on beads or on activated fixed platelets (AFP), i.e. in a system free of platelet signaling and secretion mechanisms.
U.S. Pat. No. 5,399,667 “Thrombospondin receptor binding peptides” by Frazier et al., teaches novel short peptides that bind to the thrombospondin 1 receptor, which preferably have five amino acid residues which share the tetrapeptide Arg-Val-Ala-Val (SEQ ID NO: 20) and have the specific sequences. The patent further teaches a VVM-containing peptide that binds to the thrombospondin 1 receptor selected from the group consisting of RFYVVMWKQVTQS (SEQ ID NO: 8) (Seq ID No. 1 in '667) and fragments thereof containing at least the sequence SEQ ID NO: 3, and SEQ ID NO: 4 and fragments thereof containing at least the sequence SEQ ID NO: 5.
U.S. Pat. No. 5,190,920 “Method for using synthetic analogs of thrombospondin for inhibiting metastasis activity” to Jacob et al. relates generally to peptide fragments and synthetic analogs of thrombospondin (TSP) which retain thrombospondin-like activity. Compounds and compositions comprising fragments and methods for using synthetic analogs of thrombospondin for promoting or inhibiting thrombospondin-like activity are provided
PCT Publication WO 1996/040033 to Thaddeus et al. describes a hemostatic patch composed of a biodegradable matrix, epsilon aminocaproic acids (EACAs), and thrombin receptor activating peptides (TRAPs). The invention discloses many representative embodiments containing gelatin, alginates, oxidized regenerated cellulose, or collagen as a matrix, and EACAs, TRAPs, calcium, RGD peptide, and calcium as active components. Disclosed sequences include SFLLRNPNDKYEPF (SEQ ID NO: 9), SFLLRNPNDKYEP (SEQ ID NO: 10), SFLLRNPNDKYE (SEQ ID NO: 11), SFLLRNPNDKY (SEQ ID NO: 12), SFLLRNPNDK (SEQ ID NO: 13), SFLLRNPND (SEQ ID NO: 14), SFLLRNPN (SEQ ID NO: 15), SFLLRNP (SEQ ID NO: 16), SFLLRN (SEQ ID NO: 17), SFLLR (SEQ ID NO: 18), SFLL (SEQ ID NO: 19), SFL and their derivatives.
U.S. Pat. No. 7,285,580, “Methods of using primer molecules for enhancing the mechanical performance of tissue adhesives and sealants” to Stedronsky teaches that in addition to the natural proteins various recombinantly-produced proteins may also find use in tissue adhesives and sealants. Not only may the natural proteins described above be recombinantly produced, but also various crosslinkable non-natural recombinant proteins will find use herein. Preferred non-natural recombinantly produced proteins include proteins which comprise repeating units of naturally occurring amino acid sequence blocks from such naturally occurring structural proteins as fibroin, elastin, collagen, keratin, and the like. Preferred repetitive unit proteins for use include SELPOK, SELPOK-CS1 and SELPOK.
PCT Publication WO 2009/040034 “USE OF A PEPTIDE AS A THERAPEUTIC AGENT” to Bevec et al. is directed to the use of the peptide compound Arg-Phe-Tyr-Val-Val-Met-Trp-Lys-OH (SEQ ID NO: 1) as a therapeutic agent for the prophylaxis and/or treatment of cancer, autoimmune diseases, fibrotic diseases, inflammatory diseases, neurodegenerative diseases, infectious diseases, lung diseases, heart and vascular diseases and metabolic diseases and further relates to pharmaceutical compositions preferably in form of a lyophilisate or liquid buffer solution or artificial mother milk formulation or mother milk substitute containing the peptide Arg-Phe-Tyr-Val-Val-Met-Trp-Lys-OH (SEQ ID NO: 1) optionally together with at least one pharmaceutically acceptable carrier, cryoprotectant, lyoprotectant, excipient and/or diluent.